摘要
甲基汞是重要的全球性污染物和持续性有机污染物(POP),具有强神经毒性,胎幼儿脑组织对其神经毒性作用尤为敏感。为了解长期低剂量接触甲基汞对中枢神经系统发育的影响,本研究建立了长期接触氯化甲基汞致大鼠脑发育损伤实验动物模型,采用毒理学,分子生物学和蛋白质组学技术研究了甲基汞暴露对大鼠仔鼠脑发育的影响及发育过程中蛋白质差异表达。研究发现:孕期及哺乳期甲基汞暴露可影响大鼠仔鼠的脑发育,仔鼠脑汞含量明显升高,神经元凋亡率增加。甲基汞暴露可造成仔鼠脑发育过程中蛋白的差异表达,出现差异表达蛋白中有9种蛋白参与体内能量代谢、线粒体功能和信号传导过程,与甲基汞神经毒性作用密切相关。本研究为甲基汞神经毒作用分子机制的研究和预防提供理论依据,具有重要的理论和现实意义。
Methylmercury is an important neural toxic substance and continuously organic pollutant, and have caused several poisonous accidents historically. There have many researches on methlmercury toxicity worldwide, however the mechanism still remains unclear. Methylmercury mainly affects on central neural system, developmental brain is more sensetive to it’s neurotoxicity. Our study aims at investigating damages to developmental rat brain and possible mechanism undergoing with toxicology, molecular biology and proteomics techniques.
一、Effects of chronic MMC exposure on developmental brain of rat
1.Establishment of rat brain injured model by chronic MMC exposure Female Wistar rats (90±10g) were randomly assigned to the experimental groups, dietary containing various amounts of MMC and one control group without MMC. The experimental groups were fed standard rat chow with 1.0mg MMC (ExpⅠ) ,2.0mg MMC (ExpⅡ) and 3.0 mg MMC (ExpⅢ) respectively for 90 days before gestation to 30d post parturition.
2.Toxicity and reproductive toxicity by chronic MMC exposure Based on statistical analysis, compared with control group, the weight, pregnant rate, the number of pups of rats in ExpⅡwere reduced (p<0.05), however significant decrease were observed in ExpⅢ(P<0.01). Pups of different experimental group were normal in the appearance.
3.Analysis of Hg concentrations in rat brain
Pups from each group were euthanized by decapitation on postnatal days (PND) PND1, 7and 30. Whole brain was removed immediately. Brain samples were numbered and weighed accurately on an electronic scale, then used for analysis of Hg concentrations by cold atom absorption Hg2+ concentration analysis apparatus. The results showed: The concentrations of Hg2+ in pups’brain of experimental groups at various postnatal days were markedly higher than those of the control group (p<0.05). Time-course studies in experimental group showed the concentrations of Hg2+ in pup’s brain increased gradually during development and reached its peak at PND30. The results indicated that MMC could across blood-brain barrier which accumulated in pup’s brain. The enhanced effect was dose-dependent.
4. Investigation on morphology of developing rat brain injured by MMC
Paraffin sections of brain samples were made of each group using standard methodology and stained with HE, then examined by light microscope. Apoptotic changes revealed cell shrinkage and nuclear pyknosis these were in the part of the neurons of the cerebra and hippocampus and were especially apparent in the Pukinje neurons of cerebellum.
5. Functional manifestations of neurotoxicity during rat early development caused by chronic MMC exposure
Forearm hanging time of pups in ExpⅡ(2.0mg/kg) and ExpⅢ(3.0 mg/kg)were significant shorter than that in control group . compared the CNS developmental indicator of pups in ExpⅡand ExpⅢwith that of control group , there was Significant difference on rate of surface righting, cliff avoiding and air righting. No difference of that in ExpI(p>0.05)。
6. Apoptosis of neuron during development induced by MMC
Changes of programmed cell death (PCD) rate of neuron from cerebellum and hippocampus caused by chronic MMC exposure were detected using ApoAlertTM DNA Fragmentation Assay Kit. The results showed that apoptosis were seen in developing neurons from both control and experimental groups, however the PCD rates in various experimental groups were significantly higher than that of control group accompanied with the increasing concentration of Hg2+ (p<0.05). These results indicated there exists a relationship between damage in the developing brain and excessive apoptosis of neurons induced by MMC.
二、Identification and relative-proteomic analysis on the developmental neurotoxicity of methylmercury
1 . Preliminarily establish and optimize the two-dimensional gel electrophoresisin (2-DE) and computer image analysis way with the proteomic research for the developmental neurotoxicity of methylmercury.
The Immobilized pH gradient was used as ID.Some applications, such as sample preparation and volume of loading sample, was used aschoice of IPG gel were improved. Sliver staining, ImageMaster 2D 5,0 analysis software.
Sliver staining, ImageMaster 2D 5,0 analysis software from Neurotoxicity of methylmercury . Repetitive experiments shows that in three experiments in a row, the amount of protein spots of the same team sample deviates from the relative standard as following (the average of variation coefficient %) :neurotoxicity of methylmercury group and the normal group is 23.00±10.11 and 20.33±9.90 respectively. (the range of variation coefficient %):Neurotoxicity of methylmercury and the normal group is 3.80-6.89 and 2.70 -6.89 respectively. The isoelectric point and molecular weigh of the same protein spots in three experiments deviates from relative standard as following: 8.93%±1.17%,10.16%±2.02% and 10.87%±3.86% respectively. Therefore, better resolution and repetitive 2-DE atlas were obtained. The optimized 2-DE and image analysis way is a better method to support further study of developmental neurotoxicity of methylmercury proteomeic.
2.Protein expression differences of rat hippocampus detected by 2-DE.
Protein expression difference of protein extracts from high methylmercury treated group were analyzed by 2-DE technique. Compared with control postnatal 1 day group , among twenty differential expression pots detected, expression level of thirteen proteins was up-regulated , whereas the other seven protein expression was down-regulated. More than 500 protein spots were also detected in postnatal 30 days group on electrophoresis maps of both by using silver staining method. A total of 39 protein spots showed significant differential expression between the MMC exposure group and control goup (P < 0.01).
3.Identification of differential expression protein.
Protein spots were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). The nine identified proteins including: Mn-SOD, annexin1,PGM-1, cyclophilin A, cofilin1,profilin,SPARC, GFAP, OPN, could be divided into several categories according to their functions such as: energy-metabolism; mitochondrion function; signal transduction ;protein metabolism ;nucleotide synthesis and other proteins etc.
Our study primarily testify that MMC exposure could affect rat brain development, cause developmental brain damage and differential expression of protein. The possible mechanism of neurotoxicity by MMC may relate to interruption of energy-metabolism, mitochondrion function and signal transduction process. However, the exact mechanisms need to be further investigated.
引文
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